心肌α肌球蛋白重链启动子驱动绿色荧光蛋白表达载体构建及鉴.docVIP

心肌α肌球蛋白重链启动子驱动绿色荧光蛋白表达载体构建及鉴 　 关键词】 心作者：潘国栋 黄永章， 郭凌郧， 唐俊明， 孔 霞， YANG Qinglin， 王家宁 肌特异性；肌球蛋白重链；启动子；增强型绿色荧光蛋白；干细胞 [摘 要] 目的： 构建并鉴定心肌α肌球蛋白重链启动子驱动的绿色荧光蛋白表达载体（MHC-EGFP），该基因只在心肌中特异性表达。 方法： 设计5和3分别具有SalI/HindⅢ酶切位点的EGFP引物，聚合酶链反应法（PCR）从pLEGFP质粒中扩增增强型绿色荧光蛋白（EGFP）的cDNA，插入pGEM-T Easy载体表达盒，构建pGEM-EGFP，SalI/HindⅢ双酶切后回收729 kb片断，插入经相同酶切的含5.5 kb心肌特异性α肌球蛋白重链启动子的pNC26质粒，构建pMHC-EGFP。连接产物转化感受态DH5α，挑选克隆，PCR扩增鉴定，重组质粒经NotI酶切，回收7.2 kb MHC-EGFP表达盒。分离培养小鼠心肌细胞与骨骼肌成肌细胞。MHC-EGFP表达盒通过脂质体法分别转染心肌与骨骼肌成肌细胞。荧光显微镜观察转染细胞是否出现绿色荧光，以鉴定MHC-EGFP表达盒是否具有表达特异性。结果： EGFP cDNA正确插入pNC26质粒中αMHC启动子3端。成功转染MHC-EGFP表达盒的心肌细胞出现绿色荧光，而转染的骨骼肌成肌细胞无荧光出现。结论： MHC-EGFP表达盒具有心肌特异性表达特性，为进一步监测干细胞向心肌分化研究奠定基础。 [关键词] 心肌特异性；肌球蛋白重链；启动子；增强型绿色荧光蛋白；干细胞 Abstract: Objective To construct and identify the cardiac -myosin heavy chain promoter (MHC) -driven enhanced green fluorescent protein (EGFP) expression vector (pMHC-EGFP), which solely expresses EGFP in cardiomyocytes. Methods EGFP cDNA primers were synthesized with forward primer and reverse primer containing SalI and HindⅢ, respectively. EGFP cDNA was amplified by polymerase chain reaction (PCR), added single deoxyadenosine at 3’ ends, and subcloned into pGEM-T easy vector to construct pGEM-EGFP. The fragment of 729 bp from SalI/HindⅢ-digested pGEM-EGFP was subcloned into pNC26, a plasmid carried 5.5 kb MHC promoter, to construct pMHC-EGFP. The expression cassette of 7.2 kb MHC-EGFP was recovered with low melting point agarose electrophoresis. Mouse cardiomyocytes and skeletal muscle myoblasts were isolated and cultured in vitro. MHC-EGFP expression cassette was transfected into cardiomyocytes and myoblasts, respectively. EGFP expression was observed under fluorescent microscope. Results EGFP cDNA was successfully inserted into the 3? ends of MHC promoter. The green fluorescence could be observed in transfected cardiomyocytes, but not in transfected myoblasts. Conclusion MHC-EGFP expression cassette can be specifically expressed within cardiomyocytes, which provides a basis to monitor